*Protein ID (NP or XP #) or Wolbachia#: LmjF14.1370
*Organism (including strain): Leishmania major
Etiologic Risk Group (see link below): Risk Group 2
*Background/Disease Information (sort of like the Intro to your Mini Research Write up): Leishmania major is a protozoa that causes cutaneous leishmaniasis, which harms the macrophages and dendritic immune cells of the host. It is transmitted by the female sandfly and can cause uncomfortable skin lesions. Leishmaniasis is quickly transmitted from person to person in sandy regions, which are hotspots for sandflies. (The disease may also be transferred through the sharing of needles, but this occurs in only a small minority of cases.) When a sandfly sucks the blood of an infected individual, it takes in the Leishmania and becomes a carrier of the disease. Leishmaniasis is transmitted when sandflies carrying the Leishmania parasite engage in a subsequent blood meal, injecting the young parasite, or promastigote, into the flesh wound of the human. Macrophages of the host engulf the Leishmania promastigote, which matures inside of the host cell and proceeds to infect other macrophages.
Essentiality of this protein:
Gene/Ortholog: Tb927.7.3620 (OG4_11028); Phenotype: significant gain of fitness in procyclic forms; Source study: alsford
Complex of proteins?: yes
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): Homo sapiens (1)
Synthetic analogues of SB-219383. Novel C-glycosyl peptides as inhibitors of tyrosyl tRNA synthetase. (2001).
--Show screenshot of BRENDA enzyme mechanism schematic
Fig 1. Schematic of reaction mechanims of phosphorylation of L-tyrosine by tyrosyl-tRNA synthetase in L. major.
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): The effect of substrate binding on trypanosomal tyrosyl-tRNA synthetase was assayed by adding substrate or other potential ligands to 0.5 mg/ml protein in standard buffer and monitoring fluorescence over a temperature range of 20–90° C. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3095712/
Sigma Aldrich Supplier
ATP disodium salt hydrate-----$ 37.20 (1mg)-----FLAAS
L-tyrosine-----$65.50 (50g)----- T3754 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC390865/pdf/pnas00363-0045.pdf
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 3P0J
Show pairwise alignment of your BLASTP search in NCBI against the PDB
-- Max % Identities: 100%
-- % Positives: 100%
Current Inhibitors: N/A
Expression Information (has it been expressed in bacterial cells): Yes, expressed in E. coli.
*NCBI Gene # or RefSeq#:
*Protein ID (NP or XP #) or Wolbachia#: LmjF14.1370
*Organism (including strain): Leishmania major
Etiologic Risk Group (see link below): Risk Group 2
*Background/Disease Information (sort of like the Intro to your Mini Research Write up): Leishmania major is a protozoa that causes cutaneous leishmaniasis, which harms the macrophages and dendritic immune cells of the host. It is transmitted by the female sandfly and can cause uncomfortable skin lesions. Leishmaniasis is quickly transmitted from person to person in sandy regions, which are hotspots for sandflies. (The disease may also be transferred through the sharing of needles, but this occurs in only a small minority of cases.) When a sandfly sucks the blood of an infected individual, it takes in the Leishmania and becomes a carrier of the disease. Leishmaniasis is transmitted when sandflies carrying the Leishmania parasite engage in a subsequent blood meal, injecting the young parasite, or promastigote, into the flesh wound of the human. Macrophages of the host engulf the Leishmania promastigote, which matures inside of the host cell and proceeds to infect other macrophages.
Link to TDR Targets page (if present): http://tdrtargets.org/targets/view?gene_id=27213
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): http://tritrypdb.org/tritrypdb/showRecord.do?name=GeneRecordClasses.GeneRecordClass&primary_key=LmjF14.1370
Essentiality of this protein:
Gene/Ortholog: Tb927.7.3620 (OG4_11028); Phenotype: significant gain of fitness in procyclic forms; Source study: alsford
Complex of proteins?: yes
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): Homo sapiens (1)
Synthetic analogues of SB-219383. Novel C-glycosyl peptides as inhibitors of tyrosyl tRNA synthetase. (2001).
Bioorg Med Chem Lett 11: 711-4.
*EC#: 6.1.1.1
Link to BRENDA EC# page: http://brenda-enzymes.org/php/result_flat.php4?ecno=6.1.1.1
--Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): The effect of substrate binding on trypanosomal tyrosyl-tRNA synthetase was assayed by adding substrate or other potential ligands to 0.5 mg/ml protein in standard buffer and monitoring fluorescence over a temperature range of 20–90° C.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3095712/
Sigma Aldrich Supplier
ATP disodium salt hydrate-----$ 37.20 (1mg)-----FLAAS
L-tyrosine-----$65.50 (50g)----- T3754
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC390865/pdf/pnas00363-0045.pdf
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 3P0J
Show pairwise alignment of your BLASTP search in NCBI against the PDB
-- Max % Identities: 100%
-- % Positives: 100%
Current Inhibitors: N/A
Expression Information (has it been expressed in bacterial cells): Yes, expressed in E. coli.
Purification Method: Ni-NTA affinity chromatography
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MNTDERYKLLRSVGEECIQESELRNLIEKKPLIRCYDGFEPSGRMHIAQGIFKAVNVNKC
TAAGCEFVFWVADWFALMNDKVGGELEKIRIVGRYLIEVWKAAGMDMDKVLFLWSSEEIT
SHADTYWRMVLDIGRQNTIARIKKCCTIMGKTEGTLTAAQVLYPLMQCCDIFFLKADICQ
LGLDQRKVNMLAREYCDLIGRKLKPVILSHHMLAGLKQGQAKMSKSDPDSAIFMEDTEED
VARKIRQAYCPRVKQSASAITDDGAPVATDDRNPVLDYFQCVVYARPGAAATIDGTTYAT
YEDLEQAFVSDEVSEDALKSCLIDEVNALLEPVRQHFASNEEAHELLEAVKSYRKDGATL
PLAETALPAAPAKPHACMWMPALLKVPLDVAEGMIKVTKDFIAAHPEGTVTLVLPDWSAV
ASDEITGVEKDISAALQVNCALLKAYGLPSSVKIVTENEVILGNCDDFWVSVIGIARKNL
LSHVEELYGGEVRNAGQVIAALMRVATALMLSVSHVISTSLDGHINAFAREYTKERIDCV
QTLEGRIPALHRPGAAPAVLGADDVLYLDDNDMDIRRKIKKAYSAPNEEANPVISVAQHL
LAQHGALSIERGEANGGNVSYNTPEALVADCGSGALHPADLKAAVLQLLLDRSAQARALL
NGELKKNMTVLRNAEKKMAKKR
*length of your protein in Amino Acids: 682aa
Molecular Weight of your protein in kiloDaltons using the **Expasy ProtParam** website: 74.949 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 71945
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only): ATGAACACGGACGAGCGCTACAAGCTCCTCCGCAGCGTCGGAGAGGAGTGCATTCAGGAA
AGTGAGCTGCGCAACCTTATCGAAAAGAAGCCGCTCATTCGCTGCTACGATGGCTTCGAG
CCCTCCGGCCGCATGCACATTGCTCAGGGTATCTTCAAGGCAGTCAATGTCAACAAGTGC
ACCGCCGCCGGGTGCGAGTTTGTCTTCTGGGTGGCGGACTGGTTTGCGCTCATGAACGAC
AAGGTCGGCGGCGAACTGGAGAAGATCCGCATTGTTGGCAGGTACCTTATTGAGGTGTGG
AAGGCGGCGGGCATGGACATGGACAAGGTTTTGTTTTTATGGAGCAGCGAGGAGATCACG
AGCCACGCCGACACGTACTGGCGCATGGTGCTCGACATTGGCCGCCAGAATACGATCGCC
CGCATCAAGAAGTGCTGCACCATCATGGGCAAGACGGAAGGCACGCTGACGGCGGCACAG
GTGCTGTACCCGCTGATGCAGTGCTGCGACATCTTCTTTCTCAAGGCCGACATCTGCCAG
CTGGGCCTGGACCAGCGCAAGGTGAACATGCTCGCACGCGAGTACTGCGATCTGATCGGC
CGCAAGTTGAAGCCGGTCATTCTGTCACACCACATGCTGGCCGGTCTGAAGCAGGGGCAG
GCGAAGATGAGCAAGAGTGACCCGGACAGTGCCATCTTCATGGAAGACACCGAGGAGGAT
GTGGCACGTAAGATCCGTCAGGCCTACTGTCCGCGCGTCAAGCAGTCCGCCAGCGCCATC
ACAGATGACGGGGCGCCGGTCGCCACGGACGACCGCAACCCGGTGCTGGACTACTTCCAG
TGTGTCGTCTACGCCCGACCCGGCGCTGCGGCCACCATTGACGGCACCACCTACGCGACC
TACGAGGACCTCGAGCAGGCTTTTGTGAGCGACGAGGTCAGCGAGGATGCCCTCAAGTCG
TGCCTGATTGATGAGGTGAACGCCCTGCTGGAGCCCGTGCGCCAACATTTTGCGTCAAAC
GAGGAGGCTCACGAGCTGCTGGAGGCGGTGAAGTCGTACCGTAAAGATGGCGCGACCCTT
CCACTGGCCGAGACAGCGCTCCCTGCCGCCCCCGCAAAGCCGCACGCGTGCATGTGGATG
CCGGCGCTGCTGAAGGTGCCGCTGGATGTCGCGGAAGGGATGATCAAAGTCACCAAGGAC
TTCATCGCCGCCCACCCGGAGGGCACGGTGACACTGGTGCTGCCTGACTGGTCGGCGGTG
GCCAGCGACGAAATCACCGGGGTGGAGAAAGACATCTCGGCCGCCCTCCAGGTGAACTGT
GCGCTTCTCAAGGCGTATGGACTGCCGAGCTCGGTGAAGATCGTGACGGAGAACGAGGTC
ATTCTCGGCAACTGCGACGACTTCTGGGTGAGTGTAATCGGCATTGCACGCAAGAACCTG
CTGAGCCACGTCGAGGAGCTGTACGGGGGAGAGGTACGCAACGCTGGTCAGGTCATTGCT
GCCCTCATGCGCGTTGCCACGGCTCTGATGCTATCCGTCTCGCACGTCATCTCAACCTCG
CTGGATGGACACATCAACGCCTTTGCCCGCGAGTACACGAAGGAGCGGATTGACTGCGTG
CAGACGCTGGAGGGACGCATCCCGGCGCTGCACCGCCCTGGCGCCGCCCCAGCGGTGCTC
GGTGCCGACGACGTCCTATACCTGGACGACAATGACATGGACATCCGCCGTAAAATTAAG
AAGGCGTACTCGGCGCCGAACGAGGAGGCCAATCCGGTCATATCGGTTGCCCAGCACCTT
CTCGCACAGCATGGAGCCCTCAGCATCGAGCGCGGGGAGGCCAACGGTGGCAACGTAAGC
TACAACACGCCAGAGGCCCTCGTGGCAGACTGCGGCAGCGGCGCTCTCCACCCTGCCGAC
CTCAAGGCGGCGGTGCTACAGCTGCTGCTGGACCGGTCGGCGCAGGCGCGCGCGCTGCTC
AACGGCGAGCTCAAGAAGAATATGACCGTTCTTCGCAACGCGGAGAAGAAGATGGCAAAG
AAGAGGTAA
*GC% Content for gene: 61.44%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
ATGAACACAG ATGAGCGCTA CAAGCTCCTG CGTTCCGTTG GTGAGGAGTG CATCCAGGAG TCCGAGCTGC GCAATCTCAT TGAGAAGAAG CCTCTGATCC GCTGCTATGA TGGTTTCGAG CCCAGCGGCC GCATGCACAT CGCCCAGGGG ATCTTCAAGG CCGTGAACGT CAACAAATGC ACCGCGGCGG GTTGCGAATT CGTGTTCTGG GTCGCGGACT GGTTTGCCCT TATGAACGAT AAGGTGGGCG GCGAGCTGGA GAAGATTCGC ATCGTGGGGC GTTACCTTAT CGAGGTGTGG AAGGCGGCGG GCATGGACAT GGATAAGGTG CTGTTTCTGT GGTCCTCTGA GGAGATCACC TCGCACGCCG ACACCTACTG GCGTATGGTT CTCGATATCG GCCGCCAGAA CACAATCGCA CGGATCAAGA AGTGTTGCAC GATCATGGGC AAGACGGAAG GCACACTCAC TGCTGCCCAG GTGCTGTACC CGTTGATGCA GTGCTGCGAC ATCTTTTTCT TGAAGGCGGA TATCTGCCAG CTGGGCCTTG ACCAACGTAA GGTGAACATG CTCGCCCGCG AGTACTGTGA CCTCATTGGC CGCAAGCTTA AGCCCGTGAT TTTGTCCCAT CATATGCTCG CCGGGCTTAA GCAGGGCCAG GCGAAGATGT CGAAGAGCGA TCCCGACTCG GCGATTTTCA TGGAGGACAC GGAGGAGGAC GTGGCCCGGA AGATCCGCCA GGCCTACTGC CCCCGCGTGA AGCAGTCCGC GTCGGCCATC ACTGATGACG GCGCGCCGGT GGCGACAGAT GACCGCAACC CGGTGCTGGA CTATTTCCAG TGCGTGGTGT ACGCGCGCCC CGGTGCGGCG GCCACGATCG ACGGCACTAC GTACGCAACG TACGAGGACC TGGAGCAGGC GTTCGTTAGC GACGAGGTGA GCGAGGACGC GCTCAAGTCG TGCCTGATCG ATGAGGTCAA CGCACTGCTC GAGCCCGTCC GGCAGCACTT CGCCTCTAAC GAGGAAGCGC ATGAGCTGCT GGAGGCCGTG AAAAGCTACC GGAAGGACGG TGCGACACTC CCGCTGGCCG AGACGGCGCT CCCGGCGGCC CCCGCTAAGC CGCACGCGTG CATGTGGATG CCGGCCCTTC TGAAGGTGCC GCTGGATGTT GCAGAAGGTA TGATCAAGGT GACTAAGGAC TTCATCGCAG CGCACCCTGA AGGCACAGTG ACGCTGGTCC TCCCTGATTG GTCCGCCGTG GCGAGCGACG AGATCACCGG CGTCGAGAAG GATATCAGCG CGGCTCTGCA AGTTAATTGC GCTCTCCTCA AGGCGTACGG CCTGCCATCC AGCGTGAAGA TCGTGACAGA GAATGAGGTC ATCCTGGGCA ACTGCGACGA CTTCTGGGTC TCTGTCATCG GGATTGCACG CAAGAACCTG CTCAGCCATG TGGAGGAACT GTACGGCGGC GAGGTGCGCA ACGCCGGCCA GGTGATCGCG GCACTGATGC GCGTGGCCAC AGCCCTGATG CTGAGCGTCA GCCATGTTAT TTCTACCTCT CTGGACGGCC ACATTAATGC CTTCGCGCGC GAATACACGA AGGAGCGCAT CGACTGCGTG CAGACGCTGG AGGGTCGTAT CCCGGCGCTG CACCGGCCCG GCGCCGCGCC TGCCGTGCTC GGCGCGGACG ACGTGTTGTA CCTGGACGAC AACGACATGG ACATCCGTCG CAAGATCAAG AAGGCCTACA GCGCTCCGAA CGAGGAAGCC AACCCCGTCA TCTCCGTGGC CCAGCATCTC CTGGCGCAAC ATGGCGCGCT GTCCATTGAG CGCGGGGAGG CCAACGGCGG CAACGTGTCG TACAACACGC CGGAGGCCCT GGTGGCGGAC TGCGGCAGCG GTGCGCTCCA CCCCGCCGAT CTCAAGGCTG CGGTGCTCCA GCTTTTGCTG GATCGTTCTG CACAGGCTCG TGCGCTCCTC AACGGTGAGC TGAAGAAGAA TATGACGGTG CTTCGCAATG CCGAGAAGAA GATGGCGAAG AAGCGCTAA
*GC% Content for gene (codon optimized): 61.2 %
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):